The goal of this proposal is to continue the development of a synthetic live bacterial therapeutic forhomocystinuria, an inborn metabolic disorder leading to accumulation of the amino acid methionine, and resultsin dramatically increased risk of stroke and other thrombotic conditions. Petri Bio's approach will be capable ofbreaking down methionine in the gut to reduce or eliminate dependence on a methionine restricted diet andresult in decreased plasma and tissue homocysteine resulting in superior clinical outcomes. The condition isestimated to occur at an incidence of 1 in 250,000, however some reports indicate a potential incidence of 1 in65,000 when accounting for the current imprecise diagnostic assays and often subtle symptoms which mayevade clinical detection until they become severe or obvious, such as lens detachment from the center of theeye. Cystathionine beta-synthase (CBS), the gene mutated in classical homocystinuria, is localized at a keyregulatory branch point in the eukaryotic methionine cycle. CBS catalyzes a pyridoxal 5"²-phosphate dependentbeta-replacement reaction condensing serine and homocysteine (Hcy) into cystathionine that is subsequentlyconverted to cysteine in a reaction catalyzed by cystathionine-γ-lyase (CGL, EC 4.4.1.1). Inactivation of CBS bymutation results in classical homocystinuria (HCU) which in human subjects, is characterized by a range ofconnective tissue disturbances including marfanoid habitus and lens dislocation, intellectual impairment, and adramatically increased incidence of vascular disorders particularly thromboembolic complications such as stroke.Treatment strategies for pyridoxine non-responsive HCU typically attempt to lower plasma and tissue levels ofHcy by a combination of restricting dietary intake of the Hcy precursor methionine and dietary supplementationwith trimethylglycine, more commonly referred to as betaine. Petri Bio, Inc. has developed a novel strategy forenzyme therapy, employing prokaryotic strains compatible with the human gut microbiome to serve asexpression vectors for therapeutic protein administration. After in silico screening of bacterially-derivedmethionases for a number of desirable characteristics of therapeutic enzymes, ten have been cloned, expressed,and shown to reduce methionine concentrations in vitro. During this Phase I program, we will extend thesestudies by screening hundreds of bacterially-derived methionases in silico and subsequently cloning, expressing,and testing in vitro methionine catalysis capabilities. In vitro tests will be undertaken to measure methionaseactivity of these bacterial strains. After optimization of strains with high methionase activity, we will evaluate theirability to reduce methionine concentrations in vivo, as well as ameliorate the effects of methionine accumulationin a murine model of HCU. Future studies will optimize the bacterial methionase transgenes to ensure maximumactivity and biocompatibility as well as select a lead candidate bacterial strain for preclinical drug development.
Public Health Relevance Statement: Project Narrative
Classical Homocystinuria (HCU) is a rare metabolic condition caused by mutations in the cystathionine-
β-synthase (CBS) gene. Unlike other rare metabolic genetic diseases, the enzyme is not an ideal candidate for
direct enzyme replacement therapy as its clinical safety and efficacy has not been demonstrated and in vitro
testing indicates a risk of tumorigenesis. A major barrier to current treatment is adherence to a methionine
restricted diet. Petri Bio, Inc. has developed a novel strategy for producing enzyme therapeutics expressed in
bacterial vectors compatible with the gut microbiome. This approach allows excess metabolites (such as
methionine in HCU) to be reduced in the gut prior to systemic uptake and thus mimic the effect of a low
methionine diet without actually eating one. Here we will evaluate proof-of-concept in vivo efficacy of this
approach against HCU in a transgenic murine model of the disease.
Project Terms: Accounting ; Amino Acids ; aminoacid ; Betaine ; Lycine ; Oxyneurine ; Western Blotting ; Western Immunoblotting ; protein blotting ; Catalysis ; Cloning ; Connective Tissue ; Cystathionine ; Cystathionine beta-Synthase ; Cystathionine Synthetase ; Cystathionine β-Synthase ; Serine Sulfhydrase ; beta-Thionase ; methylcysteine synthase ; Cysteine ; Half-Cystine ; L-Cysteine ; Diet ; diets ; Disease ; Disorder ; Eating ; Food Intake ; Enzymes ; Enzyme Gene ; Escherichia coli ; E coli ; E. coli ; Exhibits ; Future ; Genes ; Goals ; Homocysteine ; Homocystinuria ; cystathionine synthase deficiency ; Human ; Modern Man ; In Vitro ; Incidence ; Lens dislocation ; Lyase ; Lyase Gene ; Metabolic Diseases ; Metabolic Disorder ; Thesaurismosis ; metabolism disorder ; Methionine ; Methods ; Mutation ; Genetic Alteration ; Genetic Change ; Genetic defect ; genome mutation ; Plasma ; Blood Plasma ; Plasma Serum ; Reticuloendothelial System, Serum, Plasma ; Production ; Pyridoxal Phosphate ; Pyridoxal 5-Phosphate ; Recombinant Proteins ; Risk ; Safety ; Serine ; L-Serine ; Stroke ; Apoplexy ; Brain Vascular Accident ; Cerebral Stroke ; Cerebrovascular Apoplexy ; Cerebrovascular Stroke ; brain attack ; cerebral vascular accident ; cerebrovascular accident ; Surveys ; Survey Instrument ; Testing ; Tissues ; Body Tissues ; Vascular Diseases ; Vascular Disorder ; blood vessel disorder ; vascular dysfunction ; vasculopathy ; Measures ; Vitamin B6 ; Pyridoxin ; Pyridoxol ; VIT B6 ; Vitamin B 6 ; pyridoxine ; base ; human subject ; Clinical ; Phase ; Ensure ; Dietary Supplementation ; diet supplementation ; uptake ; Transgenes ; Therapeutic ; Metabolic ; programs ; diet restriction ; restricted diet ; dietary restriction ; Dependence ; Reaction ; gastrointestinal ; biocompatibility ; biomaterial compatibility ; enzyme therapy ; success ; Protein Replacement Therapy ; enzyme replacement therapy ; expression vector ; microbial ; transgenic ; Transgenic Organisms ; Toxicities ; Toxic effect ; disorder model ; Disease model ; Reporting ; Modeling ; Property ; native protein drug ; pharmaceutical protein ; protein drug agent ; therapeutic protein ; therapeutic enzyme ; Dietary intake ; Oncogenesis ; tumorigenesis ; Symptoms ; Adherence ; Data ; Detection ; Ph.D. ; PhD ; Doctor of Philosophy ; Pre-clinical Drug Testing/Development ; Preclinical Drug Testing/Development ; pre-clinical drug development ; Preclinical Drug Development ; Mutate ; in vitro Assay ; in vivo ; Characteristics ; Development ; developmental ; Human Microbiome ; human-associated microbiome ; vector ; novel strategies ; new approaches ; novel approaches ; novel strategy ; Outcome ; Impairment ; bacterial vector ; bacteria vector ; mouse model ; murine model ; eye center ; lens ; lenses ; treatment strategy ; in vitro testing ; efficacy testing ; screening ; diagnostic assay ; gut microbiome ; GI microbiome ; digestive tract microbiome ; enteric microbiome ; gastrointestinal microbiome ; gut-associated microbiome ; intestinal biome ; intestinal microbiome ; Genetic Diseases ; genetic condition ; genetic disorder ; lead candidate ; stroke risk ; risk for stroke ; risk of stroke ; in silico ; thrombotic ; thrombotic complications ; thromboembolic complications ; thrombosis complications ;
